ibs, can be a diagnostic “nut” - norsk sykepleierforbund lsomhet og ibs v... · faecal...

No defined biomarker for IBS

Diagnosis by well defined clinical features.....?- Manning criteria 1978

- Rome I and Rome II 1999, at least 12 weeks or more duringthe last year have experienced abdominal pain/discomfort that have

2/3 features

* Relief with defecation* Change in frequency of stools* Change in form (appearance) of stools

IBS, can be a diagnostic “nut” .

Gut 2000;47:506

Dyspepsia

Irritable bowel syndrome

Carbohydrateintolerance

IBD

Affect approx 12-15% of the population in EU and USA (India 25%)

About 40% of the IBS pts seek medical advice from family physician

- 12 % of all primary care consultations

- 40% referred for colonoscopy

Typical work-up in a single patient; $ 2387 = € 2000

Cost the American health system $ 25 000 000 000 !!!

IBS, a significant economic burden to the society.

IBS and health cost in 8 European countries

UK, Fr, Ge, Sp, It, Ne, Be, Su = 330 mill people

- Total cost US-$ 40 000 000 000

- 1/3 is direct cost like colonoscopy etc

- 2/3 Indirect cost, absenteeism, disability

What does IBS cost in the EU countries ?

Quigley Dig Liver Dis 2006

Alarm symptoms like rectal bleeding in young persons; what does it really mean ??

20 % of the population will have rectal bleeding in a year

Only 50% of CRC do actually bleed, and if so usually intermittently………..

A study from the USA, showed that you will have to do

11500 scopes to diagnose one CRC in a patient below

40 years of age …………

The incidence of Colorectal cancer by different age groups in Norway

The average incidence between 2004-2008

Total 3535 (m/f: 1756 / 1779)

Total < 50 years of age 10%



- Average waiting list for colonoscopy in the UK is 28 weeks

- Kings college implemented F-Cpt in clinical practice 5 years ago

- They pre-screen all younger (< 45) referred for colonoscopy,a negative test leads to “no colonoscopy”

- “Waiting list” for colonoscopy in the UK, 28 weeks, Kings College, 8 days

Prof. Bjarnasson, pers com.

Faecal Calprotectin in Irritable Bowel Syndrome;“the London experience”

Faecal Calprotectin in Irritable Bowel Syndrome in Sweden and the UK 2010

The use of laboratory analysis in SwedenQuality and cost-effectiveness in test utilizationPhD thesis from University of Uppsala 2010Mirja Mindemark

“F-Calprotectin screening could lead to cost avoidance of € 17-23 mill annually in Sweden as compared to direct referral for colonoscopy”

National Health Service UK 2010, purchasing and supply service.

Economic reportValue of Calprotectin in screening out IBSFebruary 2010The use of Calprotectin in the detection of inflammation of the bowel has the potential to improve the management of IBS pts in primary care, reducing the needfor referral to Secondary care. Health care recourses utilisation would therefore bereduced.

Case story l

40 year old woman, many years with abd. pain and diarrhea.

Normal colonscopy and gastroscopy including biopsies

Normal Calprotectin

Weaping during both examinations, despite sedation…?

Follow-up consultation, sexually abused by close relativeand living in a violent marriage

Needs a psychiatrist not a gastroenterologist

Data from the USA, shows that among women with IBS, as many as

22- 50 % have been neglected or sexually abused during childhood.

When balloon- stimulating the rectum, using 50 ml balloon, they

experience strong pain.

Functional MRI of the brain shown enhanced activity in the central

region of the brain

Ringel Gastroenterology 2008

Ringel Gastroenterology 2008

Functional MRI in an abused IBS patients

Malabsorption in post-infectious IBS patients

Case story l l

32 year old woman meny years troubbled with abd. pain and diarrhea.

At age 16 diagnosed with IBS……………?

2010, Calprotectin < 1250mg/kg (n< 50)

Telephone conversation offering colonoscopy next day; ” no thank you,On my way to Tenerife”. It´s only my IBS, I´m used to that.

Colonoscopy 3 weeks later; massive Crohns disease of the colon andT. Ileum

Both parents were physicians…….

116 pts with IBS according to the Rome ll criteria

93 women and 23 men

All pts had a sigmoidoscopy (< 40) or a colonoscopy (>40) w/biopsy

4 weeks diet eliminating; - cow milk protein- wheat protein- egg- tomato- chocolate

Pts improved, underwent a double blind, placebo controlled food challenge

Food intolerance and irritable bowel syndrome

Carroccio Clin Gastro Hepatol 2010

DBPC was performed administrating capsules containing- milk protein

* casein* lactalbumin* lactglobulin

- wheat protein

Serum Total IgE and allergen specific IgE was assessed- egg- cow milk- soy- peanut- wheat- tomato

Food intolerance and IBS, examinations I


Flow Cytometric Allergen Stimulation Test (Flow2Cast)

Patients were tested on the following allergens:- a- lactalbumin

- b- lactalbumin

- casein

- egg white and yolk

- wheat

- soybean

- fish

- tomato

- plus others if indicated

Intra assay and inter assay variation was 2.5% and 6,8%

Reproducibility, 20 pts and 3 investigators; Kappa value 0.92

Food intolerance and IBS, examinations II


Food intolerance and IBS, results


44 of the pts improved during the 4 weeks food elimination period

These underwent DBPC food challenge

- 19 were intolerant for both cow milk and wheat protein

- 3 were intolerant for cow milk only

- 2 were intolerant for wheat only

Other foods causing IBS like symptoms

- egg 16 cases

- tomato 12 cases

- soy protein 5 cases

- yeast, pork, prawns, fish, celery 3 cases each

Patients and controls (non-IBS) were tested using

- Flow2Cast vs total IgE, p< 0.0001

Cow milk

allergy

- Flow2Cast vs sp-IgE p< 0.01

- Flow2Cast vs total IgE p< 0.0001

Wheat allergy

- Flow2cast vs sp-IgE p< 0.01

Food intolerance and IBS, results from BAT


Food intolerance and IBS, results and

conclusion

In conclusion, in patients with IBS, this method is feasible for

making a diagnosis for food hypersensitivity.

It may replace routine allergy tests such as skin prick test and

specific IgE assays.


Results from Gluten vs placebo

Gluten

Placebo

VAS score 0= none, 100 worst

October 2009Cellular Allergy / MC

Flow2 CAST® Principle

positive control anti FceR

Allergen

pseudo allergic reaction (non IgE mediated)

anti CCR3 PE

anti CD 63 FITC/ fmlp


Method

EDTA- Blood

Allergens

Stimulation Buffer

Staining Reagent

Stimulation Control and


CCR3 as Basophil Selector

CC

R3

-PE

SSC

BasophilsEosinophils

Neutrophils

Lymphocytes

Monocytes

In conclusion,

IBS is a heterogeneous patient group, were organic

aetiology cannot be ruled out, but colonoscopy is NOT

necessary the thing to do

Cow milk allergy in children

Affects between 2 – 3 % (7%) of children < 1 year of age

Requires complete exclusion of milk protein in diet

50% of the children outgrows the hypersensitivity by 1 year,80 – 90 % by age 5.

Vandenplas Arch Dis Child 2007

Symptoms of cow milk allergy in children

Often simultaneous symptoms from many organs;- atopic dermatitis 50 – 70 %- gastrointestinal symptom 50 – 60%

* vomiting * diarrhoea* malabsorption* failure to thrive

- respiratory tract 20-30%

Dietary elimination of cow milk protein, also from mother in case of breast feeding

In case of improvement, reintroduction of CMP. If resumption of symptoms, further testing.

- Specific IgE testing (Rast), sens approx 67 % (cutoff 2kUA/l)

- Prick test, may be dangerous

- Basophile Activation Test (BAT) Flow2Cast

Diagnosis of cow milk allergy in children

Rubio Allergy 2010

Results from a comparison study

Sensitivity and specificity for detection of CMP allergy - IgE; 67% / 67%

- SPT; 100% / 21%

-BAT; 91% / 90%

Kappa statistics for oral CMP challenge- IgE 0.35

- SPT N A

- BAT 0.752

Rubio Allergy 2010;1398


Flow2 CAST® Pipetting Scheme

1. Preparation of tubes– Add 50 µl Allergen/Control (in Stimulation Buffer)

– Add 100 µl Stimulation Buffer (contains IL-3, Ca2+ and Heparin)

– Add 50 µl Whole Blood (EDTA)

– Add 20 µl Staining Reagent (contains CCR3-PE/CD63-FITC)

2. Stimulation Incubate 15’ / 30’ at 37°C in water bath 15’

3. Lyse Add 2 ml Lysing Solution 5’ at RT 5’

4. Spin 5’ at 500 x g 5’

5. Resuspend cells with 300 µl Wash Buffer

6. Measure Acquire data

7. Analyze


Patient BackgroundStimulation buffer+Blood+Staining

Positive ControlStimulation control +Blood+Staining

AllergenAllergen X +Blood+Staining

Samples to Measure

PB

PC

A1


Lysing of red blood cells

Centrifugation Analysis

Results within 45 min


• Choice between technology

• evaluated and adapted allergens

• efficient tools for non IgE mediated drug allergy diagnosis

•provide additional benefit to sIgE

BÜHLMANN Cellular Allergy Package


Advantages of the new protocolFast protocol

• Short incubation times

• Time to result 1h!

• Incubation times down to 25 minutes

Whole blood

• Easier handling

• no cell isolation

• More reproducible (leucocytes recovery)

Basophil selection with CCR3

• Easier gating and data analysis

• Two color assay (FITC and PE)

Test suitable for routine Laboratories


A major concern is the seriousness of allergic reactions

Around 30% of the western population is affected by allergies during their lifetime

A Swiss study estimates the incidence of life threatening anaphylaxis caused by Allergy at 8 -10 cases per 100000.

60% of anaphylactic shocks are caused by Hymenoptera stings

20% of anaphylactic shocks are caused by reactions to drugs.

10% of anaphylactic shocks are caused by food allergies


Bee & Wasp Venoms

1. Clarification of ambiguos results:

2. Fewer false positive and false negative results

3. Optimised differentiation between bee wasp venom allergy

4. Successful monitoring of specific immuno theraphy


Why CCR3 as Basophils detector

CC

R3

-PE

IgE-

FITC

Donor 2Donor 1 Donor 3

Total IgE: 1.9 to 2.3 IU/ml 17 to 21 IU/ml


D’après M Dvorak et al. JACI 1994

BASOPHIL

Before stimulation


D’après M Dvorak et al. JACI 1994After stimulation


Allergy is a chronic disease with high morbidity, causing high costs and has become a significant socio-economic topic in society.


Total Health cost estimates reach a level of 30 Billion Euros for Western Europe, 1.5-2

Billion CHF in Switzerland alone.


Which diagnostic measures are usually taken?

Skin tests

sIgE measurements in the patients serum


CLEAR

DIAGNOSIS

Skin Test ++

IgE ++

70-80%UNCLEAR

DIAGNOSIS

~20% 10%NEGATIVE

DIAGNOSIS

Double positive results due to

VENOM CROSS-REACTIVITY.

!Skin Test +/-

IgE +/-

Skin Test -

IgE -

allows for clearer

diagnosis!

CAST®-2000

Flow-CAST®A very high number of double positivities can

be resolved by CAST

®-Assays!

75% of undiagnosed cases can be

solved by CAST®-Assays!

1) Bee and Wasp Venoms


... AND – no other assay can identify the culprit venom so well, when double positive results occur with serology and in vivo tests are applied!

Clinical Diagnosis CAST 2000 Flow CAST CAP FEIA Skin Test

Bee+ / Wasp - 93% 83% 57% 58%

Wasp+ / Bee - 99% 90% 51% 62%

Relative Specificity of in vivo and in vitro methods in cases of double positivity:

(Scherer et al.2008)

1) Bee and Wasp Venoms


References


Food additives•CellularAllergy Assays are reliable tools for the diagnosis of non IgE mediated food additive intolerances

•Excellent sensitivities for commonly used food additives

•To optimise sensitivity the reccommended allergen combination to be tested is Benzoate, Nitrite and Salicylate


Recent publication on Flow CAST®

application in pediatrics:


Advantages of the new protocolFast protocol

• Short incubation times

• Time to result 1h!

• Incubation times down to 25 minutes

Whole blood

• Easier handling

• no cell isolation

• More reproducible (leucocytes recovery)

Basophil selection with CCR3

• Easier gating and data analysis

• Two color assay (FITC and PE)

Test suitable for routine Laboratories


IgE –mediated Allergy

Non IgE –mediated Allergy

Basophil characterization marker

Basophil activation marker

Allergy Mediators


Flow2 CAST (FK-CCR)

0

10

20

30

40

50

60

70

80

90

base pos control peanut allergen peanut allergen

%C

D63

cut off 15%

77%

2.6%

49%

46%

c1 c2

Patient with Allergy to peanut

Child with anamnesis for peanut, previous eczema, asthma. Challenge pos. for peanut. sIgE unspecifically positive for 3 more allergens additionally to peanut.

Flow2 CAST clear cut signal specifically for peanut. 46-49% of the basophil population expose CD63 on their outer membrane surface.

Negative control below 15%.

Clinical samples tested with Flow2 CAST®


New Marker Combination CCR3/CD63

ibs, can be a diagnostic “nut” - norsk sykepleierforbund lsomhet og ibs v... · faecal...

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